Effect of Sodium Acetate and Trace Element (Se²⁺, Zn²⁺) on Exopolysaccharide Production by Lactobacillus plantarum and Promote Antioxidant Capacity

Abstract

L. plantarum producing EPS plays an important role as an antioxidant, anti-proliferative, and anticancer. This study aims to increase the production of EPS by L. plantarum through modification of MRS (de Mann Rogosa Sharpe) media mixed with coconut water, treated with natrium acetate, Se, and Zn at different concentration, as well as understanding its effect on antioxidant activity. The effect of adding sodium acetate with different concentrations of 0.25, 0.50, 0.75, and 1.0% into mixed media MRS coconut water, (1:3) was studied. Fermentation experiments at different of Se2+ concentration (mM): 50; 75; 100; 125; 150; and 175, and addition of variation Zn2+ concentration (mM): 2.5; 5.0; 7.5; 10.0; 12.5; and 15.0), were carried out separately. Antioxidant potential was tested by FRAP (ferric reducing antioxidant power) and ABTS (2.2′-azinobis (3-ethyl benzatiazoline)-6-sulfonate). The results showed that the addition of sodium acetate with different concentrations showed a significant difference to the dry weight of EPS (P < 0.05). The increase in sodium acetate concentration was up to 1%, in line with the increase in EPS production by L. plantarum (g/g DW biomass). The addition of Se2+ 100 mM increased the ratio of glucose to protein content by 2.121. The value of the antioxidant activity of Fe (II) was 311.54, and the ABTS test obtained IC50 of 83.041. A separate experiment with the addition of Zn2+ in the fermentation medium of L. plantarum produced a fluctuating exopolysaccharide. The value of the antioxidant activity of Fe (II) M using the FRAP method was 275.886. The IC50 value with the ABTS method is 73.2942. Characterization of EPS from L. plantarum using FTIR (Fourier transforms infrared spectrophotometry) has hydroxyl, carboxylate, and aromatic functional groups.

1. Introduction

Lactic acid bacteria (LAB) are widely used in the manufacture of traditional fermented milk. In the dairy industry, the bacteria are also useful as a culture starter in the fermentation process. Several strains of lactic acid bacteria have important roles in health which are beneficial microflora in the intestinal tract [1, 2, 3] and capable of synthesizing exopolysaccharides (EPS) [4, 5, 6, 7]. Exopolysaccharides produced by lactic acid bacteria have been given increasing attention in recent years; it is due to their contribution to the rheology and texture of food products. In addition, EPS products, which are GRAS (generally recognized as safe), are declared safe for consumption and stable during storage [4, 5].

EPS is a polymer of high-molecular weight-reducing sugars, which are secreted by microorganisms into their external environment. This polymer has bioactivation, so it can be used for anti-viral and anti-inflammatory treatment. It has an inhibitory effect on tumor growth in vitro or in vivo [8, 9, 10]. The structure and composition of EPS is closely related to its anti-tumor biological function. In food industry, exopolysaccharides can function as thickeners, gelling agents, and emulsifiers. EPS from lactic acid bacteria (LAB) can exert functional effects on food, improve the rheology of fermented dairy products, and have beneficial health effects.

Several types of LAB that produce exopolysaccharides are Lactobacillus acidophillus, L. rhamnosus, L. casei, L. reuteri, Bifidobacterium longum, and L. plantarum [11]. The amount of exopolysaccharide produced by lactic acid bacteria is influenced by several factors, such as media composition (C, N concentration, and mineral supplementation), fermentation conditions, interactions between strains (co-culture fermentation), and fermentation technology (fed-batch fermentation). Other factors include physico-chemical conditions such as temperature, pH, level of oxygen presence, incubation time, and genetic factors [12].

Microbes need minerals to synthesize cellular components, to produce energy, and becomes electron acceptors in the metabolism of glucose and other sugars. Some minerals are enzyme activators for microbial metabolism such as Se²⁺, Zn²⁺, Mn²⁺, Mg²⁺, Ca²⁺, and others. The addition of minerals with the right concentration into the growth medium of L. plantarum will increase the formation of exopolysaccharides.

Previous research on the effect of mineral species on EPS production and growth characteristics of L. bulgaricus strain ropy in milk media showed that the best mineral source was 0.5% sodium acetate, which yielded up to 476.6 mg/L of EPS compared to triammonium citrate, potassium phosphate, and magnesium sulfate at concentrations of 0–0.5% [13]. Referring to the results, this study added the mineral sodium acetate with a concentration variation of 0.25–1.0% into coconut water media to produce EPS by L. plantarum. The results of previous studies using coconut water producing the highest EPS of 7.0510 g with a composition of 75% coconut water [14].

The addition of selenium (Se²⁺) micronutrients to MRS media was intended to review its effect on increasing antioxidant potential. Selenium as a microelement acted as a component of the enzyme glutathione peroxidase (GPX) which had antioxidant activity by reducing peroxide compounds, so it reduced free radicals in the body. Selenium is an essential function in the biological system [15]. The results of previous studies reported that the amount of selenium that could be added to bacterial culture was around 100–150 mM [16]. Micronutrient selenium became an important nutrient for cell proliferation that played its role in increasing cell growth [17]. Some LAB strains have been reported to be capable of resisting selenium oxyanions at high concentrations during cultivation. Especially, L. plantarum has been suggested as Se-enriched lactobacilli for food applications.

Research on micronutrients Zn and Cu added to goat's milk production showed an effect of increasing antioxidant activity. L. casei KCTC 3260, was found to possess a high antioxidant ability by chelating Fe²⁺ or Cu²⁺, although no detectable SOD activity was observed [18]. This paper reports the results of research on the effect of adding selenium to the growth medium of L. plantarum bacteria for the production of exopolysaccharides that have potential as antioxidants.

Another micromineral is zinc (Zn) which is important for health. Zn is needed by various organs of the body, such as the skin of the gastrointestinal mucosa. Zn increased the antioxidant capacity of SOD which played its role in protecting pancreatic beta cells from damage caused by reactive oxygen species (ROS). Superoxide is one of the most abundant ROS produced by the mitochondria, while SOD catalyzes the breakdown of superoxide into hydrogen peroxide and water and is therefore a central regulator of ROS levels [19]. The complex form of copper zinc SOD (Cu Zn-SOD) compound was able to increase the activity of SOD. Study on the L. fermentum E-3 and E-18 were able to express Mn-SOD to resist oxidative stress [20]. Increased antioxidant activity of EPS from L. plantarum culture with the addition of zinc (Zn) with various concentrations of 2.5–15 mM will be reported here. Previous researchers reported that the uptake of zinc (Zn) in lactic acid bacteria (LAB) was 10 mM [13]. The results were used as a reference. Few studies about Zn-enriched LAB have been conducted, but it has been found that the bacterial growth and probiotic effect of L. plantarum can be enhanced by zinc in the gut [21].

In the past few years, intensive research of EPS from microbes has developed and widely applied as ingredients of functional foods, pharmaceuticals, nutraceuticals, cosmetics, and insecticides. EPS from microbes are predicted to quickly develop as fast as EPS from plants and microalga which now dominate the market. This is due to their role as an immunostimulatory [22, 23], antivirus activity, antibacterial, and anticancer [23, 24, 25, 26]. The potential of EPS as antioxidant becomes crucial in the field of medicine and food industry due to their action as scavengers of reactive oxygen species (ROS). ROS are a diverse group of unstable and highly reactive oxygen-derived molecules, such as hydrogen peroxide (H₂O₂), hydroxyl radical (•OH), singlet oxygen (¹O₂), and superoxide (O₂−). These oxidants are produced under stress conditions that cause destruction of macromolecules (lipids, proteins, and DNA) and disrupt various redox signalling pathways in eukaryotic cells. The oxidative stress condition caused by the abnormally high levels of ROS triggers cardiovascular disease, and has also been implicated in diabetes, various types of cancer, neurologic and inflammatory diseases, ageing [27, 28, 29], and autoimmunological disorders [30].

Sustainable Development Goal 3 of the 2030 Agenda for Sustainable Development is to “ensure healthy lives and promoting well-being for all at all ages” [31]. One of the objectives that must be achieved is to reduce the mortality rate caused by non-communicable disease such as cardiovascular, cancer, diabetes, or chronic respiratory disease. In 2019, it was reported that globally, 74% of all mortality were caused by non-communicable diseases. In this regard, this study aims to produce EPS from L. plantarum bacteria as raw material for drugs that are useful for human health. The study focused on increasing the production of EPS through modification of MRS media mixed with coconut water by adding natrium acetate, Se, and Zn²⁺ and evaluating the potential of EPS as an antioxidant.

2. Materials and methods

2.1 Microorganism

Stock cultures of L. plantarum strains were preserved in a freezer at −80°C, in cryotubes containing 10% glycerol. The culture was stored at the Indonesian Institute of Sciences, which is located in Bogor, Indonesia. The strain was inoculated into MRS broth medium (Merck, Germany) for regeneration and incubated at 37°C.

2.2 Regeneration medium of L. plantarum

The regeneration medium used was solid MRS with the composition: MRS 5.22% w/v and agar 2% w/v. The materials were dissolved with distilled water up to 100 ml. Each was distributed as much as 4 ml into a tube and then sterilized by autoclaving at 121°C for 15 minutes with a pressure of 1 atm. The sterile medium is cooled to solidify to form slant agar.

2.3 Preculture of L. plantarum

Regenerated fresh L. plantarum bacteria were inoculated one to two loops into 25 ml of MRS broth medium; cultures were incubated at 37°C until they reached OD 1.0 as preculture.

2.4 Production of exopolysaccharides by L. plantarum bacteria

An amount of 2% precultured L. plantarum in logarithmic growth phase (OD 650 nm = ±1) was inoculated into a fermentation medium containing different microminerals to produce exopolysaccharides. Incubation was carried out at 37°C for 72 hours on a shaker incubator [32].

• MRS medium contained sodium acetate with different concentrations. The medium of fermentation used MRS 5.22% (w/v), it was dissolved in a mixture of distilled water and coconut water (with a ratio of 25:75) with the addition of sodium acetate with different concentrations, that is, 0.0; 0.25; 0.5; 0.75; and 1%.

• MRS medium contained selenium with different concentrations. The addition of Se²⁺ micronutrients to the MRS medium was 5.22% in a mixture of distilled water and coconut water (with a ratio of 25:75) was carried out with variations in concentrations of 50, 75, 100, 125, 150, and 175 mM.

• MRS medium contained zinc with different concentrations [33]. The addition of micronutrient Zn²⁺ into 5.22% MRS medium in a mixture of distilled water and coconut water (with a ratio of 25:75) was carried out with various concentrations of 2.5, 5.0, 7.5, 10, 12.5, and 15 mM.

2.5 Extraction of exopolysaccharides from L. plantarum

MRS medium modified with the addition of micronutrients was inoculated with L. plantarum bacteria at a concentration of 2% (v/v) and was cultivated at 37°C for 72 hours. Cultures of L. plantarum were boiled at 100°C for 15 minutes to inactivate enzymes that degraded exopolysaccharides. The cultures were cooled and centrifuged at 14.534×g, for 15 minutes, at 4°C, to separate the cell biomass and supernatant. The precipitated biomass was then dried and weighed as the weight of L. plantarum cell biomass (mg), while the supernatant was extracted to obtain EPSs. An amount of two times of the volume of 96% ethanol was added to the supernatant for precipitation of EPSs. The mixture was stored at 4°C for 24 hours. Then, the EPS was collected by centrifugation (11.772×g, 4°C, 20 minutes). The pellets were dissolved with distilled water. EPSs were purified by adding 15% (w/v) trichloroacetic acid (TCA) [34], then was centrifuged (20,000×g, 10 minutes, 4°C). The pellets were dried at 55°C, weighed as EPS dry weight (mg) [34].

2.6 Total sugar analysis

Total sugar content was determined by the modified phenol-sulfuric acid method using glucose as a standard [35]. An amount of 1 ml of the sample was mixed with 0.5 ml of 5% phenol solution. Then, 2.5 ml of 95% sulfuric acid was added. The sample were incubated at 25°C for 10 minutes; then stirred for 1 minute. The sample solution was re-incubated for 20 minutes at 25°C. The absorbance of each sample was measured using a spectrophotometer at a wavelength of = 490 nm. Sample control used distilled water (1 ml).

2.7 Protein analysis by the method of Lowry

The standard solution used in protein analysis was bovine serum albumin (BSA). An amount of 0.5 ml of the sample was added with 0.5 ml of 1 N NaOH, shaken, and boiled at 100°C for 20 minutes, then cooled. An amount of 0.5 ml of the mixed solution (5% Na₂CO₃, 1% CuSO₄.5 H₂O and 2% Na-K-tartrate) was added to each sample; and was shaken homogeneously. Add 0.5 ml of Folin-Ciocalteu reagent to each sample; and was shaken homogeneously and let stand for 30 minutes. The absorbance of the BSA standard solution and the test sample was measured with a UV-Vis spectrophotometer at a wavelength of 750 nm. Sample control used distilled water [36].

2.8 Functional groups of EPS analysis by Fourier-transform infrared (FT-IR) spectroscopy

Sample were prepared by grinding dried EPS (1 mg) with KBr (20 mg) in 1:20 w/w ratio and pressed into 1 mm thick pellets for measurement. FT-IR spectra were recorded on an FT-IR spectrometer (Shimadzu, Japan) in the frequency range of 4000–400 cm⁻¹ [37].

2.9 In vitro antioxidant activities of EPS

2.9.1 Ferric reducing antioxidant power measurement of EPS

Various concentrations of exopolysaccharide (0, 50, 75, 100, and 125 ppm) was blended with 1 ml of 0.2 M phosphate buffer, pH  =  6.6 and 1 ml of 1% potassium ferricyanide solution. The mixture incubated at 50 °C for 20 minutes. Then 1 ml of trichloroacetic acid (10%) was added to the reaction mixture and centrifuged at 3000 rpm for 10 minutes. Then 2.5 ml of the supernatant was mixed with distilled water (2.5 ml) and 0.5 ml of 0.1% ferric chloride solution. The absorbance of the solution was measured at 700 nm using a UV spectrophotometer. Increasing the absorption of the samples means increasing the reducing power of the samples. The blank sample was distilled water, and ascorbic acid was used as positive reference standard. The ferric ion reducing power were expressed as milligrams of ascorbic acid equivalent (AAE) per ml of EPS sample [38, 39].

2.9.2 Assay of ABTS+ radical scavenging capacity

The antioxidant activity of EPS was assay based on the radical scavenging capacity of 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS+) radical with slight modification. The maximum absorption wavelength of ABTS radical ions is 734 nm, and hence the absorbance at this particular wavelength is used to detect the concentration of ABTS radical ions. Briefly, ABTS solution (7.4 mM) was mixed with potassium persulphate solution (2.6 mM) (1:1 v/v) and was left at 20–22°C in the dark for 24 hours. The ABTS stock solution was diluted with absolute ethanol to an absorbance of 0.7 ± 0.02 at 734 nm. Then, 0.2 and 0.8 ml of ABTS working solution were mixed thoroughly. VC was used as the positive control. The absorbance of the mixture solution was determined at 734 nm after 5 minutes of incubation in the dark, using a microplate reader. The ABTS radical scavenging activity (%) was calculated using the following formula:

$$\text{Scavenging rate}\left(\%\right)=\left(\text{Ac}-\text{As}\right)/\text{Ac}\times 100$$E1

Whereas is the absorbance of the test sample and Ac is the absorbance of the control at 734 nm [40, 41, 42].

2.10 Data analysis

Data were analyzed by one way ANOVA with three replications using SPSS ver 22.0 with P = 0.05. This analysis was then followed by Duncan multiple range test.

3. Results and discussion

3.1 Effect of sodium acetate concentration on the formation of EPS L. plantarum

Exopolysaccharide was extracted from the culture of lactic acid bacteria Lactobacillus plantarum fermented in coconut water-MRS medium in a ratio (25:75) at 37°C for 72 hours. The use of coconut water with a percentage of 75% referred to previous studies because it was the best percentage to produce the exopolysaccharide L. plantarum [14]. Liquid MRS medium was used because it was a selective medium for the growth of lactic acid bacteria. Seesuriyachan's research used MRS media which was a good growth medium for lactic acid bacteria; the addition of coconut water and sucrose as a carbon source could affect the yield of exopolysaccharides. The research resulted in the production of exopolysaccharides which continued to increase, that is, from a concentration of 0–100% coconut water also contains protein, sugar, amino acids, various vitamins and minerals, so with the relatively complete nutritional content, coconut water is very potential to be used as a basic material for fermenting organic acids and as an alternative carbon source for the production of exopolysaccharides from lactic acid bacteria. Lactic acid-producing bacterium is one of the bacteria that tends to be attracted to sugar-containing habitats such as coconut water [43]. Figure 1 shows the cell morphology of L. plantarum with methylene blue dye and crude exopolysaccharide extracted from L. plantarum culture.

Figure 1.

(a) Morphology of whole cells of L. plantarum visualized by microscop 1000×. (b) EPS crude extracted from L. plantarum.

The fermentation temperature used was 37^oC, which was the optimal temperature for the growth of lactic acid bacteria. The fermentation time for 72 hours was the optimal time for harvesting exopolysaccharides for the growth of lactic acid bacteria. The supernatant obtained from the extraction was then added with cold 96% ethanol in a ratio of 1:1; then allowed to stand for 48 hours at 4^oC. The addition of cold 96% ethanol aimed to precipitate exopolysaccharides [32].

The amount of exopolysaccharide produced by lactic acid bacteria was influenced by several factors such as media composition (source and concentration of carbon and nitrogen), fermentation conditions, effects of growth media (mineral supplementation), interactions between strains (co-culture fermentation), and pharmaceutical technology (fed-batch fermentation), as well as physico-chemical conditions of bacterial growth such as temperature, pH, level of oxygen presence, incubation time, and genetic factors [12].

Table 1 shows that the production of EPS (g) per dry cell biomass (g) was increasing, in line with the addition of sodium acetate concentration in MRS medium: coconut water (75:25). A significant increase in EPS production against the control (media without sodium acetate) began to be seen at a concentration of 0.75% sodium acetate: increased to 23.81%. At 1% sodium acetate concentration, EPS production increased to 34.94%. The greater the concentration of sodium acetate used the more exopolysaccharide compounds produced. It was influenced by the addition of sodium acetate mineral as an electron acceptor in the metabolism of glucose and other sugars which helped lactic acid bacteria to produce exopolysaccharide compounds [44].

No.	Na. acetate concentration (%)	Production of EPS/cell biomass dry (g/g)
1	0	28.22^a
2	0.25	29.47^a
3	0.5	31.75^ab
4	0.75	34.94^bc
5	1.0	38.09^c

Table 1.

Dry weight of EPS L. plantarum on MRS Media: Coconut water (25:75) with variations in sodium acetate concentration. The numbers followed by the same letter are not significantly different (P < 0.05).

In the exopolysaccharide polymerization reaction, the formation of carbon chains required minerals as electron acceptors binding one monomer to others. Lactic acid bacterium was not limited to oxygen as an electron acceptor. Anaerobically, some organic components could be treated with the same purpose as electron acceptors. This case particularly happened at obligate heterofermentative LAB in the alcohol or acetate formation pathway. However, it turned out that organic electron acceptors could play an important role in homofermentative LAB in anaerobic metabolism on certain substrates [44].

Research by Pham et al. also used several types of minerals in the medium to see the activity of L. rhamnoses cells in producing exopolysaccharides. The results showed that during the initial exponential growth phase the biosynthesis of exopolysaccharides did not occur. Production occurred in a stationary phase leading to death and then the exopolysaccharide produced could be reused by microbes as a carbon source due to the presence of enzymes produced by the bacteria themselves that could degrade exopolysaccharides. Consequently, the prolongation of the incubation time decreased the production of exopolysaccharides [45].

The extraction method used could also affect the amount of exopolysaccharide produced. The heating step carried out at the beginning of the extraction was able to increase the recovery of exopolysaccharides, then increased the number of exopolysaccharides obtained. The addition of ammonium sulphate at the time of extraction aimed to precipitate proteins and separate exopolysaccharide compounds from a mixture of other compounds [45].

The production of exopolysaccharides from microbial cultures depended on several different parameters. The formation of polysaccharides was most often associated with the presence of carbohydrates and low or high temperatures. Mineral requirement was also a critical factor. Restriction of nitrogen, phosphorus or sulphur sources increased the production of exopolysaccharides; on the other hand, some researchers reported that phosphate and trace elements were essential elements for the synthesis of exopolysaccharides by Pseudomonas aeruginosa. Several types of minerals were needed by microbes as growth factors needed to form energy and compose cell components and the formation of secondary metabolites. Micro elements such as K, Ca, Mg, Cl, Fe, Mn, Co, Cu, Zn, and Mo were needed by almost all microbes. The transfer of nutrients into microbial cells could be in the form of passive diffusion, diffusion with the help of permease, active transfer or through the phosphotransferase system. Minerals were generally transferred by active transfer [44].

3.2 Effect of sodium acetate concentration on total sugar content and protein in EPS

Determination of glucose levels in exopolysaccharide samples was carried out using the phenol-sulphate method using visible light spectrophotometry. This method was chosen to determine the total sugar content in exopolysaccharides because it was commonly used to determine the total carbohydrate content of bacterial polysaccharides. The advantage of this method was that the reagents used were cheap and easy to obtain, little equipment was needed and the analysis was simple [46]. The principle of this method was that simple sugar and oligosaccharide could react with phenol in concentrated sulfuric acid to produce a stable yellowish orange color. The addition of phenol and concentrated sulfuric acid aimed to form a color complex in the sample so that it could be detected by UV-VIS spectrophotometry. The addition of concentrated sulfuric acid would produce a yellowish orange hydroxymethyl furfural compound absorbing at a wavelength of 490 nm [47]. The mechanism of the dehydration reaction of glucose to hydroxy methyl furfural can be seen in Figure 2.

Figure 2.

Schematic overview of the phenol sulfuric method for the total carbohydrate analysis [47].

Figure 3a: the correlation coefficient value was 0.9924 showing that there was a linear relationship between the concentration of the standard glucose comparison solution and the absorption at 490 nm; the increase in concentration was proportional to the increase in absorption. Figure 3b: analysis of variance (ANOVA) and DMRT test (Duncan multiple range test) showed that there was a significant difference in the addition of sodium acetate with a concentration of 0–0.75% with an increase in glucose levels at that concentration. Meanwhile, at the concentration of sodium acetate concentration of 0.75% with 1% there was no significant difference even though glucose levels in the 1% sodium acetate treatment increased. Figure 3b the highest glucose level was obtained in 1% sodium acetate sample (75.54%) and the lowest was found in 0% sodium acetate sample (29.05%).

Figure 3.

(a) Relationship of standard glucose concentration to absorption at λ490 nm and (b) effect of Na acetate concentration on glucose levels in EPS (%). The numbers followed by the same letter are not significantly different (P < 0.05).

The absorption of BSA standard solution with concentrations of 20, 40, 60, 80, and 100 ppm was measured by a spectrophotometer at a wavelength of 750 nm (39) resulting in a calibration curve with the regression line equation y = 0.0034x + 0.1492 with the correlation coefficient value obtained 0.9909 (Figure 4a).

Figure 4.

(a) Relationship of protein standard concentration (BSA) to absorption at λ750 nm and (b) effect of Na acetate concentration on protein content in EPS (%). The numbers followed by the same letter are not significantly different (P < 0.05).

Determination of protein content using the Lowry method based on two different reactions. The first reaction was the formation of copper Cu+. Under alkaline conditions formed by a solution of Na₂CO₃ in NaOH, Cu²⁺ ions formed a complex with peptide bonds reducing Cu²⁺ to Cu+. The second reaction was a reduction reaction by Folin-Ciocalteu reagent (phosphomolybdate and phosphotungstate). The Cu+ ion and the radical groups of tyrosine and tryptophan reacted with Folin's reagent to produce an unstable product that reduces molybdenum or tungsten blue. Protein would react with Folin-Ciocalteu reagent to form a complex compound giving a blue color [48].

The results of analysis of variance (ANOVA) and DMRT showed a significant increase in protein content due to treatment with sodium acetate concentration (0–0.75%). However, the results did not show a significant difference between 0.75 and 1% sodium acetate concentrations. Figure 4b shows that the highest protein content was obtained as a result of 1% sodium acetate treatment (9.70%) and the lowest was found in 0% sodium acetate treatment (7.24%).

3.3 Interpretation of IR spectrum of EPS and glucose samples

The functional groups of exopolysaccharide samples produced from L. plantarum isolates with comparison, namely glucose, were determined using an FT-IR (Fourier transform infra-red) spectrophotometer [49].

The results of the interpretation of the infrared spectrum of the exopolysaccharide sample of L. plantarum and the reference standard (glucose) are presented in Table 2. Glucose was used as a comparison because glucose is the main compound contained in the exopolysaccharide.

Sample code	Wave number (cm^−1)	Absorption peak sample (cm^−1)	Function groups
Glucose	3500–3200	3275.13	Group –OH (hydroxyl)
3000–2900	2935.66	Stretching C-H (alkanes)
1300–1000	1224.80, 1151.50, 1109.07	Vibration C–C and C-O
EPS	3500–3200	3219.19	Group –OH (hydroxyl)
1800–1600	1678.07	Group –C=O (carboxylate)
1600–1475	1539.20	Group C=C (aromatic)
1300–1000	1085.92	Vibration C–C and C-O

Table 2.

Interpretation of IR Spectrum of Lactobacillus plantarum EPS samples and glucose standard.

The results of the FT-IR analysis in Table 2 show that the exopolysaccharide and glucose samples contained IR spectra similarities indicating a typical absorption of polysaccharide compounds. Glucose showed the presence of hydroxyl groups (-OH), alkane groups (–CH), and C–C vibrations. Meanwhile, the exopolysaccharide isolated from Lactobacillus plantarum showed the presence of a hydroxyl group (-OH), a carbonyl group (–C=O) in the carboxylate and a C–C vibration. These results indicated that the extract obtained was a carbohydrate compound.

The area of 200–900 cm⁻¹ was the fingerprint area for carbohydrates and could be used for carbohydrate identification. Exopolysaccharides from L. plantarum with the addition of sodium acetate had an absorption band of 1085 cm⁻¹. Absorption bands in the area around 1080 cm⁻¹ were characteristic for carbohydrates derived from microbial biomass glucose, galactose, and mannose had absorption bands in the region of 983–1200 cm⁻¹ [50]. The sample and glucose had an absorption band in that area. Specifically, glucose was in the wave number of 1100–1124 cm⁻¹ and the exopolysaccharide sample was in the wave number of 1000–1150 cm⁻¹. It indicated the presence of sugar monomers such as glucose, galactose and mannose in the sample. Thus, the sample can be said to be a polysaccharide based on the resulting IR spectra and its similarity to glucose.

3.4 Effects concentration variations of Se²⁺ and Zn‑ in fermentation media on dry weight of cell biomass (g), EPS production (g), glucose, and protein levels (%)

Table 3 shows that the higher the concentration of Se in the medium, the higher the cell biomass formed. It was inversely proportional to the formation of EPS: the higher the selenium concentration in the medium, the lower the dry weight of EPS. The decrease in EPS yield was due to the increase in selenium concentration in the media because the selenium inhibited the metabolic process of EPS synthesis by L. plantarum [51].

No.	Treatment; Conc. of Se^2+ (mmol)	Cell biomass dry weight (g)	Crude EPS production (g) per 25 ml medium	Glucose content in EPS (%)	Protein content in EPS (%)	Ratio of glucose: Protein
1.	0	0.023^a ± 0.002	2.781^g ± 0.046	18.435^a ± 0.883	14.172^a ± 0.483	1.308
2.	50	0.041^b ± 0.003	2.631^f ± 0.022	35.200^b ± 0.975	16.916^ab ± 1.293	2.091
3.	75	0.045^bc ± 0.003	2.558^e ± 0.014	36.826^b ± 1.430	18.772^b ± 2.075	2.018
4.	100	0.052^bcd ± 0.001	2.433^d ± 0.021	42.849^c ± 1.814	20.229^b ± 1.429	2.121
5.	125	0.057^cd ± 0.001	2.343^c ± 0.037	48.345^d ± 1.729	24.687^c ± 3.455	1.980
6.	150	0.063^d ± 0.004	2.246^b ± 0.021	48.070^d ± 2.138	32.380^d ± 1.448	1.488
7.	175	0.077^e ± 0.002	2.165^a ± 0.026	55.169^e ± 2.522	35.047^d ± 2.061	1.574

Table 3.

Effects of Se²⁺ treatment with different concentrations on dry weight of cell biomass (g), EPS production (g), glucose, and protein levels (%). The numbers followed by the same letter are not significantly different (P < 0.05).

Data are means ± S.D. of three replicates.

The results of Arain et al. reported that the addition of mineral treatment could change the pH of the media which caused the inhibition of the growth of Lactobacillus bulgaricus due to the high concentration of minerals. Mineral requirements for microbial growth were only in small amounts. This statement supported that the concentration of selenium as a mineral when added at high concentrations can result in inhibition of EPS production. Selenium is essential for the progression and expression of humoral and cell-mediated immune responses. Selenium enhances the phagocytic activity of neutrophil granulocytes and macrophages, and when stimulated in myogens, increases T lymphocyte counts [52].

Zinc is involved in protein synthesis and is a regulator of involved in protein synthesis an is a regulator of synaptic activity and neuronal processes. Zinc deficiency due to the reduced ingestion of this metal causes an abnormal process in the nervous system [51]. The effect of adding Zn²⁺ to medium with different concentrations is shown in Table 4: the higher the zinc concentration in the growth medium, the higher the cell biomass produced. On the other hand, the production of exopolysaccharides tended to decrease. The inversely proportional results of cell biomass and exopolysaccharides had been carried out in a study. The yield of exopolysaccharide production was inversely proportional to cell biomass because of the need for energy support in the exopolysaccharide biosynthesis process. The limitation of energy production in cells during the formation of exopolysaccharides caused the amount of biomass to be low, because the formation of the two was interrelated. Higher exopolysaccharides were produced with lower cell growth. It was possible that the cell growth pathway was blocked and converted to an exopolysaccharide synthesis pathway [43].

No.	Treatment of Zn^2+ (mmol) conc.	Cell biomass dry weight (g)	Crude EPS production (g)	Glucose content in EPS (%)	Protein content in EPS (%)	Ratio of glucose: Protein
1.	0	0.0148^a ± 0.0004	3.9539^a ± 0.063	18.97^a ± 0.551	12.57^a ± 0.551	1.510
2.	2.5	0.0144^a ± 0.0005	3.6430^b ± 0.094	35.70^b ± 1.015	27.63^c ± 1.002	1.293
3.	5.0	0.0149^a ± 0.0004	3.8763^ab ± 0.042	46.57^c ± 0.873	38.85^e ± 1.626	1.203
4.	7.5	0.0176^b ± 0.0006	3.7469^ab ± 0.084	50.87^d ± 0.503	27.73^c ± 1.150	1.836
5.	10	0.0190^c ± 0.0007	2.9172^c ± 0.254	69.20^e ± 1.082	19.03^b ± 1.193	3.650
6.	12.5	0.0226^d ± 0.0004	2.7365^c ± 0.211	50.73^d ± 0.986	10.73^a ± 1.193	4.760
7.	15	0.0324^e ± 0.0003	2.4257^d ± 0.225	47.93^e ± 0.665	30.83^d ± .250	1.55

Table 4.

Effects of Zn²⁺ treatment with different concentrations on dry weight of cell biomass (g), EPS production (g), glucose and protein levels (%). The numbers followed by the same letter are not significantly different (P < 0.05).

Data are means ± S.D. of three replicates.

The production of exopolysaccharides obtained from lactic acid bacteria was influenced by several factors such as fermentation conditions, effects of growth media (mineral supplementation), interactions between strains (co-culture fermentation), and fermentation technology (fed-batch fermentation). Regulation of cell growth at a constant pH resulted in better exopolysaccharide yields. The acidification process occurred because the production of lactate caused the glycohydrolase enzyme to become active (pH range 5). It caused the yield of EPS to decrease due to the enzymatic process. The culture conditions and the composition of the media (not only carbon sources) affect the yield of EPS and the molecular characteristics of the biopolymer [53].

Glucose equivalent EPS levels were determined using the phenol-sulfate method. Glucose did not have a chromophore so it was supposed to be reacted with phenol-sulphate to form an orange-yellow color having a maximum absorption at 490 nm. The addition of concentrated sulfuric acid H₂SO₄ caused hydrolysis of glycosidic bonds in EPS so that furfural compounds or furfural derivatives were formed which would condense with phenol to form yellow-orange compounds. The results of the measurement of glucose uptake and concentration in EPS can be seen in Table 5. The highest average glucose level was obtained in exopolysaccharide samples with the addition of Se²⁺ 175 mM concentration reaching 55.169% and the addition of 10 mM zinc of 69.20%, while the lowest glucose level was obtained at exopolysaccharide samples in the media without the addition of Se²⁺ and Zn²⁺.

No.	Conc. Se^2+ (mmol)	Se^2+ treatment		Conc. Zn^2+ (mmol)	Zn^2+ treatment
		FRAP* (mg FeSO4/mg)	ABTS** IC50 (mg/ml)		FRAP (mg FeSO4/mg)	ABTS IC50 (mg/ml)
1.	0	0.223 ± 0.008	107.320	0	0.025 ± 0.001	117.806
2.	50	0.245 ± 0.006	146.930	2.5	0.091 ± 0.001	71.172
3.	75	0.273 ± 0.038	84.096	5.0	0.168 ± 0.004	76.993
4.	100	0.254 ± 0.030	76.944	7.5	0.331 ± 0.008	72.254
5.	125	0.276 ± 0.003	74.929	10	0.438 ± 0.029	69.122
6.	150	0.285 ± 0.005	80.660	12.5	0.471 ± 0.020	66.645
7.	175	0.289 ± 0.018	61.882	15	0.479 ± 0.035	55.535

Table 5.

Antioxidant activity test of EPS L. plantarum using FRAP and ABTS methods.

*

FRAP Data are means ± S.D. of two replicates.

**

The ABTS radical is completely reduced to this concentration accompanied by the disappearance of the green color.

Glucose levels in exopolysaccharides would increase because during fermentation, lactose would be broken down into glucose and galactose which became the main carbon sources in increasing the activity of the UDP-glucose pyro phosphorylase and UDP galactose-4-epimerase enzymes. UDP galactose-4-epimerase was a key enzyme in the formation of EPS, and the enzyme would be active when there was a sugar unit in the form of galactose as a precursor in the formation of exopolysaccharides [53].

Analysis of protein content in EPS samples was determined using the Lowry method by visible light spectrophotometry. The protein in the sample would react with copper (II) sulphate under alkaline conditions to form Cu²⁺ ions and an amino acid radical group which then reacted with Folin-Ciocalteu to produce an unstable product that reduces molybdenum or tungsten blue. The reaction produced a blue-colored complex which gives the maximum absorption at 750 nm. Protein levels would decrease because the longer fermentation means the longer the opportunity for lactic acid bacteria to degrade protein, so it caused protein levels to decrease.

The results of the calculation of the ratio of high levels of glucose to protein provided information on the optimum culture conditions for producing EPS, because glucose was a monomer of EPS. The culture produced EPS with the highest glucose: protein ratio at the concentrations of Se²⁺ 100 mMol and Zn²⁺ 12.5 mMol.

3.5 Antioxidant activity test of L. plantarum exopolysaccharide with FRAP (ferric reducing antioxidant power) and ABTS (2,2′-azinobis(3-ethylbenzatiazoline)-6-sulfonate) methods

The results of the exopolysaccharide antioxidant activity test using the FRAP method showed that exopolysaccharides were potential to be antioxidants based on their ability to reduce colorless Fe3+ TPTZ (2,4,6-tripyridyl-s-triazine) to blue Fe²⁺, so that the antioxidant power of a compound was analogous to reducing ability of the compound. Fe3+ TPTZ compounds represented oxidizing compounds that might be present in the body and could damage body cells, while samples contained antioxidants which could then reduce Fe3+ TPTZ compounds to Fe²⁺ TPTZ so that Fe3+ TPTZ compounds would not carry out reactions that damage body cells. The more the concentration of Fe3+ TPTZ reduced by the sample to Fe²⁺ TPTZ, the higher the antioxidant activity of the sample.

The reducing activity of the EPS samples was determined according to the method described by Benzie and Strain [54]. Calibration using FeSO₄ which expressed as mg Fe(II) per gram extract (Figure 5a). The antioxidant activity test using the ABTS method using vitamin E as a comparison (Figure 5b), showed an IC50 of 8.3090 (μg/ml). The value of antioxidant test results from EPS L. plantarum using the FRAP and ABTS methods are listed in Table 5.

Figure 5.

(a) Standard curve of FeSO₄.7H₂O and (b) relationship of vitamin E concentration (ppm) to % inhibition.

The results of the antioxidant activity test of EPS L. plantarum treatment with a concentration of Se²⁺ 175 mmol showed the highest FRAP value, namely 0.289 ± 0.018 mg FeSO₄/mg; and the best antiradical ABTS activity, namely IC50 61,882 mg/ml. Meanwhile, Zn²⁺ 15 mmol treatment showed the highest FRAP value, namely 0.479 ± 0.035 mg FeSO₄/mg; and the best antiradical ABTS activity, namely IC50 55,535 mg/ml. The results showed that EPS L. plantarum had very strong antioxidant potential. The principle of the ABTS method was that EPS antiradical compounds would ward off free radicals marked by the loss of blue color (decolorization) in the ABTS reagent. It was indicated by a decrease in the absorbance value of the measured sample absorption. The advantages of ABTS compared to other methods were that the test was simple and easy to repeat; and the most important thing was that it was flexible and could be used to measure the activity of both hydrophilic and lipophilic antiradicals [55].

4. Conclusions

The production of EPS derived from the group of lactic acid bacteria including L. plantarum was in great demand because it was safe for consumption. The effect of EPS to improve food texture was a strategy to reduce the use of additives. The potential of EPS to be applied in the pharmaceutical field opened up opportunities for researchers to increase EPS production through modification of the fermentation medium by adding coconut water, Na-Acetate and minerals (Se²⁺ and Zn²⁺). The increase in EPS production by L. plantarum occurred due to the addition of natrium acetate concentration of up to 1%. Natrium acetate concentration of 0.75% gave significantly different EPS results to the control. EPS production by L. plantarum was optimum at 100 mmol of Se²⁺ concentration and 12.5 mmol of Zn²⁺ concentration, based on the highest ratio of glucose: protein.

The effect of Se and Zn concentrations on the antioxidant activity of EPS using the FRAP and ABTS methods shows that the higher the concentration of these trace elements, the higher the antioxidant activity.

The addition would affect the increase in enzyme activity for the synthesis of EPS. The antioxidant activity test of the L. plantarum EPS compound showed a very strong category.

Acknowledgments

The authors are grateful to Dr. Arif Nurkanto, Head of Laboratory in Research Center for Biosystematics and Evolution, National Research and Innovation Agency (BRIN), for their valuable support to complete this study. We express our gratitude to Apt. Lisana Sidqi Aliya, S. Farm., M. Biomed., and Apt. Dr. Herdini, Lecturer at Fac. of Pharmacy-ISTN, Jakarta for suggestions during the research.

Conflict of interest

The authors declare that there are no competing interests.